Metalloproteinases (MPs) are a family of enzymes which includes type I collagenase, stromelysin, and type IV collagenase. Substrate specificity for MPs include collagen, proteoglycans, laminin, fibronectin, alpha-1 proteinase inhibitor, casein, and gelatin, many of which are found in the human cornea. These enzymes may play an important role in normal connective tissue modeling and certain pathological processes, such as keratoconus (KC). This proposal is based on the belief that MPs are functionally important proteins within the human cornea. Only type I collagenase has been studied extensively in cornea. Surprisingly, our preliminary data indicate that type IV collagenase represents the predominant MP activity. Based upon these new studies, our hypothesis is that different tissues and cell types may have different regulatory mechanisms for MP enzymes and that one of these regulatory controls is altered in KC corneas thereby leading to increased type IV collagenase activity. In normal human cornea our specific aims are to use biochemical, immunohistochemical and molecular methods to: (1) examine and compare all MP activities in cornea and sclera organ cultures; (2) determine if different proteolytic activities are a modification of the same enzyme or different enzymes; (3) examine MP biosynthetic rates and regulation in different tissue and cell types; and (4) localize MPs in different tissues. Using the same methods, the specific aims for our KC studies are to: (1) confirm KC keratocyte MP studies and investigate KC organ cultures; (2) compare normal and KC cornea biosynthetic rates and regulation of MPs; (3) determine if increased MP activities seen in KC corneas are due to abnormal amino acid sequence or abnormal post translational modification; (4) localize MPs in KC and normal corneas; and (5) determine if decreased levels of tissue inhibitor of MPs could be responsible for increased activities seen in KC. To date there has been no comprehensive investigation of MP activities in human corneas Understanding of the MPs in normal human corneas goes beyond trying to elucidate the etiology of KC. Corneal or scleral melting is an important feature of corneal ulceration, alkali burns, vitamin A deficiency related keratomalacia, scleromalacia perforans and scleral melting associated with connective tissue diseases. Our data in normal ocular tissues will provide baseline data for future studies to investigate proteolytic activities in these pathological conditions.